In this revised R21 grant, we propose to further characterize a putative adult lung mesenchmyal progenitor cell. We initially isolated such cells using a high-speed cell sorting strategy taking advantage of properties that typify mesenchymal progenitors from other sites. These properties include: 1) a Sca-1+/lin neg. surface profile 2) an ability to efflux Hoechst dye (so-called SP cells), 3) negative hematopoietic marker CD45 status, and 4) expression of primitive mesenchymal genes. Additional phenotyping indicate these cells are not related to fibrocytes, but rather display features of classical multipotent marrow mesenchymal stem cells (MSCs). These shared features include expression of similar markers, a capacity to differentiate into fat, smooth muscle, or cartilage, and proliferation in an undifferentiated state under select in vitro conditions. We, thus, hypothesize that the adult lung contains a distinct mesenchymal progenitor cell that can be isolated based on dye efflux and surface phenotype. In Aim 1 we will further define the phenotype of this cell, and we will clarify whether cells are unipotent or multipotent. We also propose a set of exploratory studies to identify candiate miRNAs that maintain cells in an undifferentiate state. Using flow cytometry, we will fractionate cells based on cell size and expression of select surface antigens and then test defined fractions for the ability to differentiate into fat, smooth muscle, and cartilage. After we elucidate the mesenchymal progenitor phenotype, we will evaluate the differentiation repertoire of single cells. To do this, individual cells will be clonally expanded and tested for their ability to differentiate along different mesenchymal cell fates. In the last part of this aim, we will use miRNA expression microarrays and real-time PCR to evaluate miRNA expression patterns in undifferentiated and differentiated cells. Aim 2 is focused on examing the engraftment potential of this putative lung mesenchymal progenitor cell in the lung in vivo. To survey engraftment, we will inject GFP+ cells into 2 distinct models that involve tissue remodeling either in the proximal (CC10-IL-9 transgenic) or distal lung (bleomcyin). In this work, we will evaluate the impact of in vitro cell population expansion on engraftment. Through this work, we hope to establish a foundation for the types of studies that characterize organ systems in which stem/progenitor cell biology is more developed. [unreadable] [unreadable] [unreadable]